This is a proposal to continue the support of a Resource center for developing cryo-electron microscopy (cryo-EM) and image analysis technology. The ultimate goal of this Resource is 3-dimensional structure determination of biological macromolecules to near atomic resolution. The activities of our Resource include technological development, core and collaborative research, service, training, technology dissemination and maintaining an advisory committee. The technological development will be focused on the improvement of our JEOL4000 intermediate voltage electron microscope to make it an operationally efficient instrument for high resolution data collection at low temperature and low dose conditions. Experimental and computational strategies will be developed to determine 3- dimensional structures in four core research projects: crotoxin complex crystal, T4 gp32*I crystal, actin bundle from Limulus sperm and human herpes simplex virus. Technically, these specimens provide opportunities for different kinds of methodology development for thin crystals, in vivo crystalline arrays and large icosahedral spherical viruses. In addition, structural information from these specimens is relevant to our basic understanding of neurotoxicity, gene regulation, cytoskeleton biology and virology. An in-house expertise on cryo-sectioning procedure will be developed that will be useful for our study of the herpes viral genome structure. All these developed procedures and facilities will be used to investigate various biologically and medically important structures in collaboration with other researchers. In this proposal, we identify twelve such probable collaborative projects. Workshops or symposia will be held annually to disseminate our Resource technology and our staff members will be available to assist outside users in cryo-EM, EM of thick sectioned materials and image processing. A group of specialists will be assembled to meet annually to evaluate the progress and directions of the Resource activities.